Artificial Antigen-Presenting Cell Topology Dictates T Cell Activation

Nanosized artificial antigen-presenting cells (aAPCs), synthetic immune cell mimics that aim to activate T cells ex or in vivo, offer an effective alternative to cellular immunotherapies. However, comprehensive studies that delineate the effect of nano-aAPC topology, including nanoparticle morphology and ligand density, are lacking. Here, we systematically studied the topological effects of polymersome-based aAPCs on T cell activation. We employed an aAPC library created from biodegradable poly(ethylene glycol)-block-poly(d,l-lactide) (PEG-PDLLA) polymersomes with spherical or tubular shape and variable sizes, which were functionalized with αCD3 and αCD28 antibodies at controlled densities. Our results indicate that high ligand density leads to enhancement in T cell activation, which can be further augmented by employing polymersomes with larger size. At low ligand density, the effect of both polymersome shape and size was more pronounced, showing that large elongated polymersomes better activate T cells compared to their spherical or smaller counterparts. This study demonstrates the capacity of polymersomes as aAPCs and highlights the role of topology for their rational design.

Fluorescence spectrometry. Fluorescence intensity measurements were performed on a Tecan Spark 10M multimode plate reader.

Nanoparticle tracking analysis (NTA).
To track the number of particles and measure their hydrodynamic size distributions, a Nanosight NS300 instrument (Malvern Panalytical) equipped with a scientific complementary metal-oxide-semiconductor (sCMOS) camera was used. The camera was mounted on an optical microscope, allowing visualization of the light scattered by the injected particles that were present in the focus of an 80 μm beam generated by a single mode laser diode with a blue laser (488 nm). Large and small aAPCs were diluted approximately 200 and 700-fold in PBS, respectively, so that the number of particles in the field of view was in the recommended range of 20-100, and a volume of 1 mL was injected into the Nanosight chamber. For all measurements 3 captures of 30 sec were recorded, with a screen gain of 9 and a camera level of 11; during the data analysis the screen gain was set to 9 with a detection threshold of 5. Particle number concentrations and surface area concentrations were determined from the area under the curve of the size distribution with number weighting or surface area weighting, respectively.

Synthesis and characterization of block copolymer poly(ethylene glycol) -poly(D,L-lactide) (PEG22-PDLLA47 3).
The synthesis of PEG22-PDLLA47 3 was performed according to our previously reported procedure [1][2][3] (synthesis scheme is outlined in Figure S2a). A round bottom flask (50 mL) equipped with a stirring bar was dried using a heat gun under vacuum, followed by three cycles of flushing with argon and evacuation. Thereafter, the flask was subjected to a constant flow of argon. Monomethoxy-PEG-OH 1 (mPEG, 0.194 g, 0.2 mmol, poly(ethylene glycol) 1K, JenKem technology, lyophilized) and D,L-Lactide 2 (DLL, 1.3 g, 9 mmol, Acros) were added to the dried flask. Subsequently, dry toluene (ca. 50 mL) was added using an argon-flushed syringe and the solvent was evaporated to dry the reagents. The rotary evaporator was flushed with an argon balloon after solvent evaporation in order to maintain an inert atmosphere after drying, and this toluene evaporation cycle was repeated once more. The dried reagents were re-dissolved in dry DCM (13 mL; [DLL monomer] = 0.7M) using an argon-flushed syringe before DBU (15 µL, 0.5 equivalents relative to the amount of mPEG macroinitiator) was added using an argon-flushed organic solvent pipette. The reaction was stirred at 25°C for 2 hours until 1 H NMR analysis showed full conversion of the D,L-lactide monomer. Subsequently, the reaction mixture was diluted with DCM (25 mL) and washed twice with 1M KHSO4, once with Milli-Q water, and once with brine (100 mL each). The organic solution was dried using Na2SO4. After evaporating most of the solvent, the concentrated copolymer solution was precipitated into ice-cold diethyl ether (200 mL). The resulting waxy solid was partially dried under argon, dissolved in dioxane, and lyophilized to yield a white powder (yield = 1.2 gram, ⁓80% recovered polymer). The synthesized copolymer was characterized using 1 H NMR, GPC and DSC to determine copolymer composition, polydispersity and glass transition temperature (Tg), respectively ( Figure S2c,e,f). 1  Synthesis and characterization of block copolymer N3-PEG24-PDLLA45 5. Using a procedure similar to the ring-opening polymerization described for PEG22-PDLLA45 3, azido-dPEG TM (24)-OH 4 (220 mg, 0.2 mmol, Iris Biotech GmbH) and D,L-lactide 2 (1300 mg, 9 mmol) were used to synthesize azido-functionalized block copolymer N3-PEG24-PDLLA45 5 (synthesis scheme is outlined in Figure S2b), yielding a white powder (yield = 1.0 g, ⁓70% recovered polymer). 1 Figure S2e). DSC analysis revealed a Tg of 18°C ( Figure  S2f). IR analysis showed absorption bands at characteristic wavenumbers for functional groups including hydroxyl, alkanes and esters. Of particular interest is the absorption band that was observed at wavenumber 2107 cm -1 , which is characteristic for the azido-functionality ( Figure S2g).
UV-Vis spectrophotometer measurements were performed to determine the degree of DBCO and fluorophore labeling on the functionalized antibodies, as well as the antibody concentration. The system was equilibrated using 2 µL of Milli-Q water, followed by 2 µL of PBS buffer as a background blank. Then, 2 µL of sample was pipetted into the spectrophotometer and the absorbance spectrum was measured. To calculate the concentrations of antibodies, DBCO, ATTO488 and AF647, the absorbance was measured at 280, 309, 504, and 651 nm, respectively. All extinction coefficients and used formulas to correct for the spectral overlap of the individual molecules and calculate the respective concentrations are described in the equations and the table below.

488, 647
Degree of labeling of DBCO, ATTO488, AF647 on antibody -Theoretical estimations for antibody conjugation. Molar ratios between the antibodies and available reactive moieties on the polymersome surface were calculated. The maximum number of antibodies that could be retained on one particle (Abmax) was theoretically estimated at 900 Abs/small polymersome and 3600 Abs/large polymersome, according to the following equation: with the minimal antibody spacing s estimated at 10 nm at complete coverage of the surface area of small and large polymersomes with diameters d of 150 and 300 nm, respectively.
Due to the relatively low number of DBCO on the antibodies (DOL≈2, to prevent crosslinking), as well as their large size (resulting in steric hindrance) a moderate to low conjugation efficiency was expected. Taking these boundaries into account, an excess initial feed of antibodies to available N3 moieties was used to reach Abmax.
Calculations of antibody density, number and conjugation efficiency. The antibody spacing (average distance between two antibodies), the antibody number per particle, and the conjugation efficiency were calculated according to Equations 1-3.
(1)  Figure S1. Formation of azido-polymersomes with controlled morphologies. To a mixture of PEG-PDLLA (95 wt.%) and N3-PEG-PDLLA (5 wt.%) block copolymers in organic solvent, H2O (50% or 33%) is added to induce self-assembly of N3-polymersomes. To create large polymersomes, the polymersome solution (50% H2O) is dialyzed to H2O to form large spheres (N3-LgS), or to 50 mM NaCl in H2O to obtain large tubes (N3-LgT) through osmotically-induced shape transformation. To create small polymersomes, the polymersome solution (33% H2O) is extruded through a 100 nm membrane, prior to dialysis to H2O or 50 mM NaCl, to form small spheres (N3-SmS) or small tubes (N3-SmT), respectively. Upon removal of the organic solvent plasticizer during dialysis, the polymersomes are trapped in their morphological structure.   Engineering method for the development of an aAPC library with control over morphology and antibody density. A library with over 60 different aAPC topologies was developed in four steps, to control morphology (SmS, SmT, LgS, LgT), functionality (monofunctional αCD3 or αCD28, or bifunctional αCD3/αCD28) and density (five ligand densities).
Step 3: Effective purification of aAPCs through centrifugation.
Step 4: Quantification of the conjugated αCD3 or/and αCD28 antibodies using a fluorescence spectroscopy microplate assay.     1.6, 0.8, 0.4, 0.2, 0.1, and 0). Quantification of the concentration conjugated (a) αCD3 for monofunctional αCD3-aAPCs, (b) αCD28 for monofunctional αCD28-aAPCs and (c) αCD3 and αCD28 for bifunctional αCD3/αCD28-aAPCs of experiments (1) and (2). Controls (LgS without N3-moieties incubated with the highest antibody concentration), indicate a relative nonspecific bound antibody concentration of approximately 10%, compared to those bound to N3-polymersome-based aAPCs, indicating that 90% of unconjugated antibodies were removed. Data represents mean ± SD (n=3). Figure S9. Antibody calibration curves used for antibody quantification using fluorescence spectroscopy. a, αCD3 calibration curve used for final quantification. b, αCD28 calibration curve used for final quantification. c, αCD3 calibration curve used for two individual experiments (1) and (2). d, αCD28 calibration curve used for two individual experiments (1) and (2). Data represents mean ± SD (n=3).      Figure S16. Flow cytometry gating strategies. a, Flow cytometry gating to determine the T cell population purity after Pan T cell isolation. Representative dot plots show gating on peripheral blood mononuclear cells (PBMCs) to exclude debris, single cells to exclude doublets, viable cells to exclude dead cells and CD2 + CD3 + cells to obtain the frequency of T cells in samples before (PBMCs) and after (enriched and depleted) isolation. b, Purity of T cell isolate. Data from three individual donors are presented as mean ± standard error (SE). c, Flow cytometry gating to quantify the co-expression of CD25 and CD69 and the binding of αCD3. Representative dot plots show gating on T cells, single cells, viable cells and CD25 + CD69 + cells to acquire the frequency of activated T cells after 24 hours of culture. Representative histogram shows fluorescence intensity of αCD3 + cells to determine the binding of the aAPCs after 6 hours of culture. d, Flow cytometry gating strategy to quantify the expression of CD25 and PD-1 and analyze proliferation of CD4 + and CD8 + T cell subsets through use of CTV™. Representative dot plots show gating on CD25 + or PD-1 + to determine T cell activation after 24 hours or 3 days, and gating on CD4 + and CD8 + subsets. CTV™ fluorescence histogram was analyzed to obtain the Division Index of CD4 + or CD8 + T cells after 3 days. Figure S17. Bifunctional aAPCs enhance T cell activation compared to monofunctional aAPCs. T cells isolated from healthy donor buffy coats were stimulated with HD bifunctional (bi) or monofunctional (mono) aAPCs or soluble antibodies at 25, 50 and 125 ng/mL αCD3 concentrations and ~50, 100 and 250 ng/mL αCD28 concentrations. Unfunctionalized polymersomes (empty; 0 ng/mL αCD3) and Dynabeads TM (bead 1:1 cell) were used as negative and positive controls, respectively. a, Normalized CD25 expression (freq.·mean fluorescence intensity; NMFI), as determined with flow cytometry after 24 hours. b, c, Fold change in (b) IL-2 or (c) IFNγ production, relative to soluble αCD3+αCD28 at 125 ng/mL αCD3, as measured with ELISA after 24 hours. d, Division Index (average number of cell divisions) of CD4 + and CD8 + T cells (mean of subsets indicated) as determined through flow cytometric analysis of CTV™ fluorescence after three days of culture. All data is represented as mean ± SE (N=3 donors). Figure S18. aAPC binding to T cells. T cells isolated from healthy donor buffy coats were stimulated with a library of aAPCs or soluble antibodies at a range of αCD3 (1-500 ng/mL) and αCD28 (~2-1,000 ng/mL) concentrations. Binding of aAPCs or soluble antibodies to T cells as determined with flow cytometry after 6 hours of culturing. a, Binding of aAPCs per polymersome morphology. b, Binding of aAPCs per density. Dotted line represents theoretical concentration at which cells should be saturated with αCD3 (~15 ng/mL, based on 124,000 CD3 molecules/T cell) 4 . All data is represented as mean ± SE (N=3 donors, n=2 replicates). Figure S19. High ligand density enhances PD-1 expression and T cell proliferation after 3 days. T cells isolated from healthy donor buffy coats were stimulated for three days with a library of bifunctional aAPCs at high (HD) or intermediate (ID) density or soluble antibodies at 25, 50, and 125 ng/mL αCD3 concentrations, and ~50, 100, and 250 ng/mL αCD28 concentrations. Ligand densities and soluble antibody were compared for small spheres (SmS), large spheres (LgS), small tubes (SmT) and large spheres (LgT). Unfunctionalized polymersomes (empty; 0 ng/mL αCD3) and Dynabeads TM (bead 1:1 cell) were taken into account as negative and positive controls, respectively. a, Normalized PD-1 expression (freq.*mean fluorescence intensity; NMFI), as determined with flow cytometry after three days of culturing. HD enhances PD-1 expression compared to ID for SmS, SmT and LgS but not LgT. b, Division Index of CD4 + and CD8 + T cells (mean of subsets indicated) as determined through flow cytometric analysis of CTV™ fluorescence after three days of culture. HD enhances the proliferation compared to ID for SmT and LgS but not SmS and LgT. All data is represented as mean ± SE (N=3 donors). Figure S20. T cell activation by Dynabeads. T cells isolated from healthy donor buffy coats were stimulated with a library of bifunctional aAPCs with different polymersome morphologies or soluble antibodies at a range of αCD3 (1-500 ng/mL) and αCD28 (~2-1,000 ng/mL) concentrations (indicated in Figures 4 and 5). Dynabeads TM (bead 1:1 cell) were taken into account as positive controls in T cell activation experiments. a, CD25 and CD69 co-expression (freq.) as determined by flow cytometry after 24 hours. b, c, Fold change in (b) IL-2 or (c) IFNγ production, relative to soluble αCD3+αCD28 at 125 ng/mL αCD3, as measured with ELISA after 24 hours. All data is represented as mean ± SE (N=3 donors, n=2 replicates).